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Biomedical subjects

S Menzo

Publications and source records attributed to S Menzo.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of R5X4 dualtropic HIV-1 primary isolates by single chemokine co-receptor ligands.

The susceptibility of HIV-1 to chemokine-mediated inhibition may be lost as a consequence of the expanded usage of chemokine co-receptors frequently occurring in clade B isolates obtained from individuals with advanced disease. Since chemokine-based immune intervention is under intense investigation, it is crucial to determine its potential effect on primary dualtropic HIV isolates characterized by simultaneous utilization of CCR5 and CXCR4 chemokine co-receptors (R5X4 viruses). In the present study, the CCR5 binding chemokine regulated upon activation normal T cell expressed and secreted (RANTES) strongly inhibited the replication of two of eight primary R5X4 viruses in mitogen-activated primary peripheral blood mononuclear cells (PBMC). The CXCR4 antagonist AMD3100 efficiently suppressed the replication of other two HIV isolates, whereas the remaining four viruses were partially inhibited by treatment with either RANTES or AMD3100. The potency of chemokine-mediated inhibition was influenced by PBMC donor variability, but it was usually independent from the levels of expression of CCR5 or CXCR4. Dual co-receptor usage was maintained by the viruses after two serial passages on U87.CD4 astrocytic cell lines expressing exclusively either CCR5 or CXCR4. The gp120 env variable domains were sequenced before and after passages on U87.CD4 cells. Virus replication into U87.CD4-CXCR4 cells did not result in changes in the V3 region but perturbed the dominant env V4 sequence. Interestingly, double passage onto U87.CD4-CXCR4 cells determined the loss of susceptibility to RANTES inhibition. In conclusion, interference with CCR5 may efficiently inhibit the replication of at least some dualtropic HIV-1 strains, whereas forced CXCR4 usage may result in viral escape from CCR5-dependent inhibitory effects.

Amino Acid Sequence↗
PubMed2001

Identification of six putative novel human papillomaviruses (HPV) and characterization of candidate HPV type 87.

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

Genome, Viral↗
PubMed2000

Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy.

OBJECTIVE: To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). DESIGN AND METHODS: A non-replicative HIV-1 molecular vector (designated pdelta prodelta env) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pdelta prodelta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). RESULTS: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P = 0.0038 and P = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P = 0.022; ritonavir, P = 0.0466) were observed. CONCLUSION: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.

Chimera↗
PubMed2000

Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis.

OBJECTIVE: To contribute to clarifying the controversy on the association between Human Herpesviruses 6 and 7 (HHV-6, HHV-7) and multiple sclerosis (MS) studying patients with relapsing-remitting MS (RRMS) with or without evidence of disease activity (clinically or radiologically evaluated). MATERIAL AND METHODS: In 25 RRMS patients, 7 suspected MS patients and 9 patients with other neurological diseases, the following parameters were analysed: i) antibody titers (IgM and IgG) against HHV-6 by indirect immunofluorescence both in serum and cerebrospinal fluid (CSF) samples; ii) PCR-detection of HHV-6 DNA and HHV-7 DNA in CSF and HHV-6 DNA in peripheral blood mononuclear cells (PBMCs). MS patients in remission underwent a gadolinium-enhanced magnetic resonance imaging in proximity of sample collections. RESULTS: No viral DNA was found in any CSF sample, HHV-6 DNA frequency in PBMCs of MS patients and controls was not statistically different. Antibody titers against HHV-6 were comparable to those of the general population. Some 30.4% of MS patients were seronegative to HHV6. CONCLUSION: Our data suggest that there is no relationship between HHV-6 or HHV-7 and MS.

Adult↗
PubMed1999

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution.

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.

Base Sequence↗
PubMed1998

Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors.

OBJECTIVE: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. DESIGN AND METHODS: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. RESULTS: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. CONCLUSIONS: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Amino Acid Sequence↗
PubMed1996

Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo.

The dynamics of human immunodeficiency virus type 1 (HIV-1) transcription was analyzed in vitro and in vivo by using a specific molecular approach which allows accurate quantitation of the different classes of viral mRNAs. Unspliced (US) and multiply spliced (MS) HIV-1 transcripts were assayed by competitive reverse transcription (cRT)-PCR, using a single competitor RNA bearing in tandem internally deleted sequences of both template species. Acute HIV-1 infection of primary peripheral blood mononuclear cells (PBMCs), monocytes/macrophages cells, and the A3.01 T-lymphocyte-derived cell line was studied; both classes of HIV-1 mRNAs increased exponentially (r2 > 0.98) at days 1 to 3 and 1 to 4 postinfection in HIV(IIIB)-infected A3.01 cells and PBMCs, respectively, whereas monocytes/macrophages infected with monocytotropic HIV(BaL) exhibited a linear (r2 = 0.81 to 0.94) accumulation of US and MS transcripts. Following induction of chronically infected ACH-2 cells, MS transcripts increased 2 h postinduction and peaked at 5 h (doubling time, 58 min), while at 24 h, US mRNAs increased 3,053-fold compared with basal time (doubling time, 137 min). To address the biopathological significance of HIV-1 expression pattern during infection progression, pilot cross-sectional and longitudinal analyses were carried out with samples from untreated and treated HIV-1-infected patients. In almost all untreated (recently infected, long-term nonprogressor, and progressor) patients, MS transcript levels followed the general trend of systemic HIV-1 activity. In patients under treatment with powerful antiretroviral compounds, viral MS transcripts rapidly fell to undetectable levels, indicating that in vivo, levels of MS mRNAs in PBMCs are closely associated with the number of newly infected cells and suggesting a new role for the quantitative analysis of HIV-1 transcription in infected patients.

Cell Line↗
PubMed1995

Studies in subjects with long-term nonprogressive human immunodeficiency virus infection.

BACKGROUND: In a small percentage of persons infected with human immunodeficiency virus type 1 (HIV-1), there is no progression of disease and CD4+ T-cell counts remain stable for many years. Studies of the histopathological, virologic, and immunologic characteristics of these persons may provide insight into the pathogenic mechanisms that lead to HIV disease and the protective mechanisms that prevent progression to overt disease. METHODS AND RESULTS: We studied 15 subjects with long-term nonprogressive HIV infection and 18 subjects with progressive HIV disease. Nonprogressive infection was defined as seven or more years of documented HIV infection, with more than 600 CD4+ T cells per cubic millimeter, no antiretroviral therapy, and no HIV-related disease. Lymph nodes from the subjects with nonprogressive infection had significantly fewer of the hyperplastic features, and none of the involuted features, characteristic of nodes from subjects with progressive disease. Plasma levels of HIV-1 RNA and the viral burden in peripheral-blood mononuclear cells were both significantly lower in the subjects with nonprogressive infection than in those with progressive disease (P = 0.003 and P = 0.015, respectively). HIV could not be isolated from the plasma of the former, who also had significantly higher titers of neutralizing antibodies than the latter. There was viral replication, however, in the subjects with nonprogressive infection, and virus was consistently cultured from mononuclear cells from the lymph nodes. In the lymph nodes virus "trapping" varied with the degree of formation of germinal centers, and few cells expressing virus were found by in situ hybridization. HIV-specific cytotoxic activity was detected in all seven subjects with nonprogressive infection who were tested. CONCLUSIONS: In persons who remain free of disease for many years despite HIV infection the viral load is low, but viral replication persists. Lymph-node architecture and immune function appear to remain intact.

Adult↗
PubMed1995

Quantitative molecular methods in virology.

During the past few years, significant technical effort was made to develop molecular methods for the absolute quantitation of nucleic acids in biological samples. In virology, semi-quantitative and quantitative techniques of different principle, complexity, and reliability were designed, optimized, and applied in basic and clinical researches. The principal data obtained in successful pilot applications in vivo are reported in this paper and show the real usefulness of these methods to understand more details of the natural history of viral diseases and to monitor specific anti-viral treatments in real time. Theoretical considerations and practical applications indicate that the competitive polymerase chain reaction (cPCR) and competitive reverse-transcription PCR (cRT-PCR) assay systems share several advantages over other quantitative molecular methodologies, thus suggesting that these techniques are the methods of choice for the absolute quantitation of viral nucleic acids present in low amounts in biological samples. Although minor obstacles to a wide use of these quantitative methods in clinical virology still remain, further technical evolution is possible, thus making the quantitative procedures easier and apt to routine applications.

DNA, Viral↗
PubMed1995

Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds.

Methods for the absolute quantitation of nucleic acids present in small amounts in biological samples (competitive PCR and competitive reverse transcription PCR) were applied to the direct monitoring of specific anti-human immunodeficiency virus type 1 (HIV-1) therapy. With these techniques, different parameters of HIV-1 activity (including genomic RNA copy numbers in plasma, proviral and late transcript copy numbers in peripheral blood lymphocytes, and mean transcriptional activity per each HIV-1 provirus) were monitored during therapy with azidothymidine or ddI. In most of these treated patients, a direct response to the antiretroviral compounds employed was detected during the first few weeks of treatment, as documented by a fast decrease of all molecular indexes of HIV-1 activity. However, residual viral replication (albeit at minimal levels) was documented during therapy in all subjects monitored in this study. In a minority of the patients under study (3 of 12), the drug-dependent viral inhibition was maintained throughout the observation time (213 to 791 days), but in 9 patients a rebound in viremia level was detected during therapy with competitive reverse transcription PCR. Sequencing analysis of a portion of the HIV-1 gene pol from cell-free virions showed that circulating viral variants bearing at least two mutations compatible with azidothymidine or ddI resistance were detectable in the patients who exhibited a rebound in cell-free HIV-1 genomic RNA copy numbers in plasma but not in one patient who maintained (for 455 days) lowered levels of viral load during ddI treatment.

Amino Acid Sequence↗
PubMed1994

Competitive polymerase chain reaction for quantitating feline immunodeficiency virus load in infected cat tissues.

To quantitate FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensure almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretic bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with the Pisa-M2 isolate of FIV.

Animals↗
PubMed1994

Competitive polymerase chain reaction and analysis of viral activity at the molecular level.

Due to the high sensitivity level (which can be pushed to the limit of one molecule) and its extraordinary flexibility, the polymerase chain reaction (PCR) is the method of choice for the detection of nucleic acids present in very low concentration in biological samples. Since the qualitative features of PCR amplification have limited its use, several PCR-based approaches for the quantitation of low-abundance nucleic acid species have been planned and proposed in the last few years. Recently, different lines of evidence have indicated that competitive PCR and competitive reverse-transcription-PCR share several advantages over other quantitative approaches. This evidence opens up unexpected possibilities in many biological fields, including virology; in fact, availability of reliable techniques for the absolute quantitation of DNA and RNA species may be the key to a better understanding of the pathogenic steps of most viral diseases and for a more precise monitoring of patients treated with specific antiviral compounds. In this review article, we summarize the procedures adopted for this quantitative molecular approach; additionally, several important technical aspects to plan novel competitive PCR-based applications are analyzed, and early results obtained using cPCR for the direct evaluation of viral activity in vivo are discussed.

DNA, Viral↗
PubMed1994

Quantitation of hepatitis C virus genome molecules in plasma samples.

A competitive reverse transcription PCR (cRT-PCR)-based assay for the quantitative detection of hepatitis C virus (HCV) viremia was developed, optimized, and applied to the direct molecular analysis of clinical samples from nine patients with persistent HCV infection. As for other competitive PCR-based applications, this method consists of the reverse transcription and subsequent amplification of two RNA species in the same tube: the wild-type template (to be quantified) and a known amount of a modified synthetic template. These templates have identical primer recognition sites and very similar (but not identical) sizes, thus allowing direct detection of both template species after gel electrophoresis and ethidium bromide staining. The results obtained by this cRT-PCR application for testing clinical samples from HCV-infected patients mainly indicate that the competitive approach reaches the degree of sensitivity (fewer than 5 HCV RNA molecules per 100 microliters) necessary to evaluate viral load in all HCV-infected patients, independently of clinical conditions, and that this technique is flexible enough to quantify highly divergent levels of cell-free HCV genome copy numbers in biological samples. Interestingly, we observed a sample-to-sample variation in the loss of detectable HCV genome molecules in serum in comparison with that in plasma from the same patient, thus indicating that serum specimens, although widely used in the past few years for qualitative molecular investigation of HCV-infected patients, cannot be used to obtain reliable quantitative data on HCV viremia from these patients.

Artifacts↗
PubMed1994

Dynamics of molecular parameters of human immunodeficiency virus type 1 activity in vivo.

The dynamics of viral activity during different phases of human immunodeficiency virus type 1 (HIV-1) infection were investigated by competitive PCR methods. In particular, we studied the time course of three quantitative molecular parameters of viral activity (genomic RNA copy number in plasma and provirus and late HIV-1 transcript molecule copy numbers in peripheral blood CD4+ T lymphocytes) in untreated patients and patients treated with specific anti-HIV-1 compounds. The results shown here indicate that direct RNA parameters are quantitative molecular indices sensitive enough to be used for a more accurate evaluation of the natural history of this infection and that an indirect parameter, the mean transcriptional activity for each provirus in CD4+ T lymphocytes, may be important in studying this infection in vivo at the molecular level. A dramatic decrease of the indices was evident at seroconversion, but the quantitative values were virtually stable throughout the time the untreated patients were studied during the clinical latency phase. Furthermore, the results indicate that an early response to antiretroviral compounds is detected in most subjects as a decrease in the viral activity level.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Vaginal micropapillary lesions are not related to human papillomavirus infection: in situ hybridization and polymerase chain reaction detection techniques.

The objective of this study was to assess the human papillomavirus DNA presence in vaginal papillary lesions, with particular regard to micropapillomatosis to better define their clinical significance. Prospective study: the study population was composed of 62 women who were recruited consecutively from the Colposcopy Centre of the Ancona University, Department of Obstetrics and Gynecology, on the grounds of vaginal papillomatosis or/and typical acuminata warts. Biopsies for routine histology, and for human papillomavirus (HPV) DNA detection by means of in situ hybridization and polymerase chain reaction (PCR) were taken from the papillary lesions and from 24 healthy women, who were selected as controls. Macroscopically, vaginal micropapillomatosis was ascertained in 51 cases (82.3%), while in 11 cases (17.7%) the colposcopic diagnosis was condyloma acuminatum. During in situ hybridization, HPV DNA positivity was observed in 8 (9.4%) out of 85 samples of squamous papillae and in 11 (64.7%) out of 17 samples of condylomata; in control specimens, HPV DNA was detected in 2 (8.3%) out of 24 bioptic samples. The correspondence between in situ hybridization and PCR was 96.1%, with 17.4% more diagnosis obtained by PCR. Vaginal micropapillomatosis may be regarded as a variation in the normal anatomy of the lower genital tract without any significant relationship with HPV infection, and as a lesion easily distinguishable from condylomata acuminata by clinical examination alone.

Adolescent↗